Endothelial Slc35a1 Deficiency Causes Loss of LSEC Identity and Exacerbates Neonatal Lipid Deposition in the Liver in Mice.

BACKGROUND & AIMS: The functional maturation of the liver largely occurs after birth. In the early stages of life, the liver of a newborn encounters enormous high-fat metabolic stress caused by the consumption of breast milk. It is unclear how the maturing liver adapts to high lipid metabolism. Liver sinusoidal endothelial cells (LSECs) play a fundamental role in establishing liver vasculature and are decorated with many glycoproteins on their surface. The Slc35a1 gene encodes a cytidine-5'-monophosphate (CMP)-sialic acid transporter responsible for transporting CMP-sialic acids between the cytoplasm and the Golgi apparatus for protein sialylation. This study aimed to determine whether endothelial sialylation plays a role in hepatic vasculogenesis and functional maturation. METHODS: Endothelial-specific Slc35a1 knockout mice were generated. Liver tissues were collected for histologic analysis, lipidomic profiling, RNA sequencing, confocal immunofluorescence, and immunoblot analyses. RESULTS: Endothelial Slc35a1-deficient mice exhibited excessive neonatal hepatic lipid deposition, severe liver damage, and high mortality. Endothelial deletion of Slc35a1 led to sinusoidal capillarization and disrupted hepatic zonation. Mechanistically, vascular endothelial growth factor receptor 2 (VEGFR2) in LSECs was desialylated and VEGFR2 signaling was enhanced in Slc35a1-deficient mice. Inhibition of VEGFR2 signaling by SU5416 alleviated lipid deposition and restored hepatic vasculature in Slc35a1-deficient mice. CONCLUSIONS: Our findings suggest that sialylation of LSECs is critical for maintaining hepatic vascular development and lipid homeostasis. Targeting VEGFR2 signaling may be a new strategy to prevent liver disorders associated with abnormal vasculature and lipid deposition.

Novel GNE missense variants impair de novo sialylation and cause defective angiogenesis in the developing brain in mice.

ABSTRACT: Glucosamine (UDP-N-acetyl)-2-epimerase and N-acetylmannosamine (ManNAc) kinase (GNE) is a cytosolic enzyme in de novo sialic acid biosynthesis. Congenital deficiency of GNE causes an autosomal recessive genetic disorder associated with hereditary inclusion body myopathy and macrothrombocytopenia. Here, we report a pediatric patient with severe macrothrombocytopenia carrying 2 novel GNE missense variants, c.1781G>A (p.Cys594Tyr, hereafter, C594Y) and c.2204C>G (p.Pro735Arg, hereafter, P735R). To investigate the biological significance of these variants in vivo, we generated a mouse model carrying the P735R mutation. Mice with homozygous P735R mutations exhibited cerebral hemorrhages as early as embryonic day 11 (E11), which subsequently progressed to large hemorrhages in the brain and spinal cord, and died between E11.5 and E12.5. Defective angiogenesis such as distended vascular sprouts were found in neural tissues and embryonic megakaryocytes were abnormally accumulated in the perineural vascular plexus in mutant mouse embryos. Furthermore, our in vitro experiments indicated that both C594Y and P735R are loss-of-function mutations with respect to de novo sialic acid biosynthesis. Overall, this study reveals a novel role for GNE-mediated de novo sialic acid biosynthesis in mouse embryonic angiogenesis.

Clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in sickle cell anemia mice.

ABSTRACT: Although it is caused by a single-nucleotide mutation in the ß-globin gene, sickle cell anemia (SCA) is a systemic disease with complex, incompletely elucidated pathologies. The mononuclear phagocyte system plays critical roles in SCA pathophysiology. However, how heterogeneous populations of hepatic macrophages contribute to SCA remains unclear. Using a combination of single-cell RNA sequencing and spatial transcriptomics via multiplexed error-robust fluorescence in situ hybridization, we identified distinct macrophage populations with diversified origins and biological functions in SCA mouse liver. We previously found that administering the von Willebrand factor (VWF)-cleaving protease ADAMTS13 alleviated vaso-occlusive episode in mice with SCA. Here, we discovered that the ADAMTS13-cleaved VWF was cleared from the circulation by a Clec4f+Marcohigh macrophage subset in a desialylation-dependent manner in the liver. In addition, sickle erythrocytes were phagocytized predominantly by Clec4f+Marcohigh macrophages. Depletion of macrophages not only abolished the protective effect of ADAMTS13 but exacerbated vaso-occlusive episode in mice with SCA. Furthermore, promoting macrophage-mediated VWF clearance reduced vaso-occlusion in SCA mice. Our study demonstrates that hepatic macrophages are important in the pathogenesis of SCA, and efficient clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in SCA mice.

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression.

Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. ß1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1-/-/Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression.

PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.

BACKGROUND: Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. METHODS: We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency. RESULTS: Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice. CONCLUSIONS: PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

Safety and efficacy of an anti-human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models.

Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

On-Chip Generation and Collectively Coherent Control of the Superposition of the Whole Family of Dicke States.

Integrated quantum photonics has recently emerged as a powerful platform for generating, manipulating, and detecting entangled photons. Multipartite entangled states lie at the heart of the quantum physics and are the key enabling resources for scalable quantum information processing. Dicke state is an important class of genuinely entangled state, which has been systematically studied in the light-matter interactions, quantum state engineering, and quantum metrology. Here, by using a silicon photonic chip, we report the generation and collectively coherent control of the entire family of four-photon Dicke states, i.e., with arbitrary excitations. We generate four entangled photons from two microresonators and coherently control them in a linear-optic quantum circuit, in which the nonlinear and linear processing are achieved in a chip-scale device. The generated photons are in telecom band, which lays the groundwork for large-scale photonic quantum technologies for multiparty networking and metrology.

Highly Sensitive, Flow Cytometry-Based Measurement of Intestinal Permeability in Models of Experimental Colitis.

BACKGROUND & AIMS: Increased intestinal permeability is seen in a variety of inflammatory conditions such as enteric infections and inflammatory bowel disease. Because barrier function can provide a key biomarker of disease severity, it often is assayed in animal models. A common methodology involves gavaging mice with fluorescein isothiocyanate-conjugated dextran (FITC-D), followed by cardiac puncture to assay plasma fluorescence on a spectrophotometer. Although the FITC-D method is relatively simple, its sensitivity is limited and enables only a single measurement because the test requires killing the subject. Herein, we describe a novel flow cytometry-based method of intestinal permeability measurement based on detection of orally gavaged ovalbumin (OVA) that leaks out of the gut. Our approach uses minute blood volumes collected from the tail vein, permitting repeated testing of the same subject at multiple time points. By comparing this assay against the gold standard FITC-D method, we show the expanded utility of our OVA assay in measuring intestinal permeability. METHODS: We directly compared our OVA assay against the FITC-D assay by co-administering both probes orally to the same animals and subsequently using their respective methodologies to measure intestinal permeability by detecting probe levels in the plasma. Permeability was assessed in mice genetically deficient in intestinal mucus production or glycosylation. In addition, wild-type mice undergoing dextran sodium sulfate-induced colitis or infected by the enteric bacterial pathogen Citrobacter rodentium also were tested. RESULTS: The OVA assay showed very high efficacy in all animal models of intestinal barrier dysfunction tested. Besides identifying intestinal barrier dysfunction in mice with impaired mucin glycosylation, the assay also allowed for repeated tracking of intestinal permeability within the same animal over time, providing data that cannot be easily acquired with other currently applied methods. CONCLUSIONS: The OVA assay is a highly sensitive and effective method of measuring intestinal permeability in mouse models of barrier dysfunction and experimental colitis.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

The barrier and beyond: Roles of intestinal mucus and mucin-type O-glycosylation in resistance and tolerance defense strategies guiding host-microbe symbiosis.

Over the past two decades, our appreciation of the gut mucus has moved from a static lubricant to a dynamic and essential component of the gut ecosystem that not only mediates the interface between host tissues and vast microbiota, but regulates how this ecosystem functions to promote mutualistic symbioses and protect from microbe-driven diseases. By delving into the complex chemistry and biology of the mucus, combined with innovative in vivo and ex vivo approaches, recent studies have revealed novel insights into the formation and function of the mucus system, the O-glycans that make up this system, and how they mediate two major host-defense strategies - resistance and tolerance - to reduce damage caused by indigenous microbes and opportunistic pathogens. This current review summarizes these findings by highlighting the emerging roles of mucus and mucin-type O-glycans in influencing host and microbial physiology with an emphasis on host defense strategies against bacteria in the gastrointestinal tract.

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C.

Hemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8-/- or F9-/- hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8-/- or PROC+/+;F9-/- mice had bleeding characteristics similar to their corresponding F8-/- or F9-/- mice. Pretreating the PROC+/+;F8-/- mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8-/- or PROC+/+;F9-/- mice in a needle puncture-induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8-/- mice but not F8-/- mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbß3 activation and decreases thrombosis in TTP.

Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbß3. However, the mechanism of GPIbα-initiated activation of αIIbß3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbß3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbß3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbß3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.

[Research Progress on the Mechanism of Tubulin in Megakaryopoiesis and Regulation of Platelet Count--Review].

Tubulin affects platelets count through the control of mitosis and the formation of pro-platelets during the maturation of megakaryoblast to platelets. Tubulin is involved in maintaining the integrity of platelet skeleton, and also participates in the change of platelet morphology during platelet activation. Some new anti-tumor drugs targeting cell mitosis are trying to reduce the effect on tubulin in order to reduce the side effect of drugs on platelet formation. In some patients with thrombocytopenia, the variation and polymorphism of the tubulin gene affect the structure of microtubule multimers, which leads to the decrease of platelet formation. This review summarized the latest progresses of tubulin in the regulation of megakaryopoiesis and thrombopoiesis.

[The latest research progress of tinnitus related treatment].

Tinnitus is a kind of phantom hearing. The quality of life of millions of people around the world is affected by it. There is no data to prove that drugs can be cured. There is no final conclusion on the clinical treatment of tinnitus. This article focuses on the treatment of tinnitus: Drug Treatment, Traditional Chinese Medicine, Surgical Treatment, Hyperbaric Oxygen Therapy, Acoustic Resonance Therapy, Transcranial Magnetic Stimulation Therapy, Cognitive Behavioral Therapy（CBT）, Tinnitus Retraining Therapy （TRT）, Multiple Compound Sound Therapy, etc. Provide more personalized tinnitus treatment programs for clinical patients.

CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice.

Rationale: Platelets play an essential role in atherosclerosis, but the underlying mechanisms remain to be addressed. This study is to investigate the role of platelets in d-flow induced vascular inflammation and the underlying mechanism. Methods: We established a disturbed blood flow (d-flow) model by partial carotid ligation (PCL) surgery using atherosclerosis-susceptible mice and wild-type mice to observe the d-flow induced platelet accumulation in the subendothelium or in the plaque by immunostaining or transmission electron microscopy. The mechanism of platelet subendothelial accumulation was further explored by specific gene knockout mice. Results: We observed presence of platelets in atherosclerotic plaques either in the atheroprone area of aortic arch or in carotid artery with d-flow using Ldlr-/- or ApoE-/- mice on high fat diet. Immunostaining showed the subendothelial accumulation of circulating platelets by d-flow in vivo. Transmission electron microscopy demonstrated the accumulation of platelets associated with monocytes in the subendothelial spaces. The subendothelial accumulation of platelet-monocyte/macrophage aggregates reached peak values at 2 days after PCL. In examining the molecules that may mediate the platelet entry, we found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) reduced the subendothelial accumulation of platelets and monocytes/macrophages by d-flow, and ameliorated plaque formation in Ldlr-/- mice on high fat diet. Supportively, CLEC-2 deficient platelets diminished their promoting effect on the migration of mouse monocyte/macrophage cell line RAW264.7. Moreover, monocyte podoplanin (PDPN), the only ligand of CLEC-2, was upregulated by d-flow, and the myeloid-specific PDPN deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages. Conclusions: Our results reveal a new CLEC-2-dependent platelet subendothelial accumulation in response to d-flow to regulate vascular inflammation.

G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Mantle Cell Lymphoma Growth in Preclinical Models.

Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin's B-cell lymphoma with poor prognosis. Despite recent advances, resistance to therapy and relapse remain significant clinical problems. G-protein-coupled estrogen receptor (GPER)-mediated estrogenic rapid signaling is implicated in the development of many cancers. However, its role in MCL is unknown. Here we report that GPER activation with selective agonist G-1 induced cell cycle arrest, DNA damage, mitochondria membrane potential abnormality, and eventually apoptosis of MCL cell lines. We found that G-1 induced DNA damage and apoptosis of MCL cells by promoting the expression of nicotinamide adenine dinucleotide phosphate oxidase and the generation of reactive oxygen species. In addition, G-1 inhibited MCL cell proliferation by inactivation of NF-κB signaling and exhibited anti-tumor functions in MCL xenografted mice. Most significantly, G-1 showed synergistic effect with ibrutinib making it a potential candidate for chemotherapy-free therapies against MCL.

L-SIGN is a receptor on liver sinusoidal endothelial cells for SARS-CoV-2 virus.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose-type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN-expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2-type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19-associated coagulopathy.